Chloride channel proteins are essential to a host of cellular functions and chloride channel dysfunction is known to be involved in the pathogenesis of a number of human disease states. We have identified and begun to characterize a family of chloride channel proteins which are structurally unrelated to other known chloride channels. One member of this family, CLIC-1, is expressed in many cell types and is very highly expressed in the apical domain of renal proximal tubule cells. Subcellular localization studies indicate that it is present in membrane compartments along the endosomal pathway. We have recently demonstrated that recombinant CLIC-1 functions as a chloride selective channel when purified from bacteria and reconstituted in phospholipid membranes. Unlike typical ion channel proteins, CLIC proteins are present in cells both in a membrane inserted form and in a soluble form in the cytoplasm. Over the past several years, a number of membrane proteins have been reported to be able to assume conformations which are soluble in aqueous solution. In some instances, it is clear that these proteins can partition between membrane-inserted and soluble states and that this partitioning is important in regulation of the activity of these proteins. We have now found that CLIC-1 is capable of inserting into phospholipid membranes from the aqueous phase and this inserted CLIC-1 can function as a chloride channel. In this grant application, we propose to study the biochemical basis and biological relevance of functional membrane insertion of this important protein.